Abstract

Three novel Gram-positive, endospore-forming bacteria were isolated from a cold and alkaline environment. Phylogenetic analysis showed that the strains were almost identical, and that they were related to Natronobacillus azotifigens 24KS-1T (95.8% identity), Paraliobacillus quinghaiensis YIM-C158T (95.1%), Paraliobacillus ryukyuensis O15-7T (94.5%), and Halolactibacillus miurensis M23-1T (93.9%). The isolates produced amylase, α-galactosidase, β-galactosidase, and β-glucuronidase, and showed optimal growth at pH 10, at 20°C, and at 2–8% (w/v) NaCl. Major fatty acids were C14:0 (10.6–11.6%), anteiso-C15:0 (25.7–32.7%), C16:1ω11c (12.2–16.0%), and C16:0 (14.0–20.4%). The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol, and meso-diaminopimelic acid was found in the cell-wall peptidoglycan. The G+C content was 38.4%. DNA–DNA hybridization between strain GCM68T and H. miurensis M23-1T was 32.4%, while hybridization to N. azotifigens 24KS-1T, Amphibacillus tropicus Z-7792T, and Paraliobacillus ryukyuensis O15-7T was below 30%. The phylogenetic analysis and G+C content place strain GCM68T in relation to species belonging to Bacillus rRNA group 1, but phylogenetic and physiologic data combined with chemotaxonomic analyses support our proposal for a new genus, Alkalilactibacillus, gen. nov., with the novel species Alkalilactibacillus ikkensis, sp. nov. (type strain is GCM68T = DSM 19937 = LMG 24405).

Notes

Acknowledgments

We acknowledge Michael Hansen for doing the SEM microscopy and Dorte Rasmussen and Karin Vestberg for technical assistance. Professor Jean Euzéby, University of Toulouse, is thanked for guidance in nomenclature. Referring to the Convention on Biological Diversity, we thank the Greenland Home Rule for permission to sample ikaite columns from the Ikka Fjord. This work was partly funded by the Villum Kann Rasmussen Foundation and the The Danish Council for Technology and Innovation.

Supplementary Figure 2. Growth of strain GCM68T at different temperatures and pH. Liquid cultures in shake flasks were inoculated with strain GCM68T and samples were withdrawn for OD600 measurements at the time intervals indicated. Growth was investigated at pH 6, 7, and 8 (not shown) and at pH, 9 (dashed lines, ♦), pH 10 (solid lines, ■) and pH 10.7 (initial pH; dotted lines, ▲). The shake flasks were incubated at 10°C, 20°C, and 25°C. The pH values at the end of the growth experiment were measured to pH 9, 10, and 10.5, respectively. All incubations were in triplicates. (DOC 380 kb)

Supplementary Figure 3. Growth rate of strain GCM68T at different temperatures and pH. Regression lines were drawn from the growth curves in Supplementary Fig. 2 in the exponential growth phase. The pH values at the end of the growth experiment were measured to pH 9, 10, and 10.5, respectively. Open bars, 10°C; solid bars, 20°C; hatched bars, 25°C. (DOC 23 kb)

Supplementary Figure 4. Growth of strain GCM68T at increasing concentrations of NaCl measured in Deep Well microtiter plates. The concentrations ranged from 0 to 25% NaCl, and growth was investigated at pH 10 at 5, 15, and 25°C. The figure shows that isolate GCM68T grows in the range from 0 to 10% NaCl with optimal growth from 2 to 8% NaCl. (DOC 28 kb)

Supplementary Figure 5. Measurement of pH in growing cultures in shake flasks. The growth media were adjusted to pH 6.0 - 8.0 using NaH2PO4/Na2HPO4, to pH 8.5 - 9.0 using NaHCO3/HCl, and to 9.4 - 11.5 using NaHCO3/Na2CO3. Samples were withdrawn for pH measurements at the time intervals indicated. (DOC 163 kb)